The multistep propagation of discrete intracellular signals allows cells to respond to cues from the extracellular environment. Among the most ubiquitous and well-studied of these are the phosphorylation cascades that culminate in the activation of mitogen-activated protein kinases (MAPKs). The enzymatic activity of MAPKs is markedly influenced by extracellular events. As a rule, p38 MAPK activity is induced by environmental stress (e.g. osmotic shock, hypoxia, heat shock, ultraviolet radiation) and pro-inflammatory stimuli and cytokines such as LPS, IL-1, TGF-beta, and TNF-alpha. The most membrane-proximal enzyme activated in the classic MAPK cascade is a serine/threonine kinase known as a MAPK kinase kinase, or MAPKKK, and the MAPKKKs that lead p38 activation include TAK1, ASK1, and MTK1 (human)/MEKK4 (mouse). Growth Arrest and DNA Damage inducible 45 (Gadd45a) was initially identified as a stress-responsive gene. Our studies of Gadd45a-deficient mice found that they died at an early age of a lupus-like autoimmune disease. Because of the known ability of Gadd45-family proteins to bind and activate MTK1/MEKK4, we asked how the absence of Gadd45a might affect p38 activation. We found that, rather than being hypoactive, p38 was spontaneously active in T-lineage cells. An in-depth analysis of this initial observation led to the following findings by our laboratory: - p38 from antigen receptor-stimulated normal T cells but not B cells robustly autophosphorylates. The autophosphorylation appeared to be on the two canonical activating residues, Thr-180 and Tyr-182. - T cell p38 activation requires Lck and Zap70 but is LAT-independent. - The TCR proximal kinases Lck, Fyn, and Zap70 phosphorylate p38 on Tyr-323, which induces autophosphorylation and enhanced activity toward other substrates. Notably, even p38 lacking Tyr-182 is activated by Tyr-323 phosphorylation. - A Tyr-323 phospho-specific antiserum detects Tyr-323-phosphorylated p38 (p-Tyr-323 p38) in T but not B cells activated via the antigen receptor. - p-Tyr-323 p38 is not detected in Lck+ Zap70- Jurkat T cells, implicating Zap70 as the effector kinase in vivo. - The alternative pathway appeared to be a major mechanism of p38 activation in T cells, because (1) in Jurkat T cells, p38 containing a Y323F substitution was poorly activated in response to anti-TCR compared to wild type (WT), and (2) dual (Thr-180/Tyr-182) phosphorylation of p38 in TCR-stimulated normal resting T cells was almost completely prevented by the p38 inhibitor SB203580, indicating that it is a consequence of autophosphorylation. - p38 from Gadd45a-deficient T cells is spontaneously phosphorylated on Tyr-323. - Gadd45a specifically binds to p38 (whether phosphorylated or not) and inhibits the activity of the p-Tyr-323 form. Importantly, Gadd45a binding does not inhibit the activity of p38 phosphorylated by MKK6 (on Thr-180/Tyr-182). - p38 phosphorylated on Tyr-323 is able to phosphorylate itself in trans; that is, one p38 molecule binds and phosphorylates another. - Auto-trans-phosphorylation involves just Thr-180 and not the canonical Tyr-182 found in the MAPK cascade. - The substrate specificity of mono-phosphorylated p38 is different from the di-phosphorylated form. This may explain why this alternative pathway has been evolutionarily conserved in T cells, because the biological effects of these two phosphorylated species would be expected to differ in vivo. - We generated p38 knock-in mice in which Tyr-323 is replaced with a Phe (p38YF). Proving the physiologic importance of the alternative pathway, TCR-mediated activation is completely incapable of activating p38 in T cells from these mice. - T cells from p38YF knockin mice are slow to transit from G0 to G1 in the cell cycle upon stimulation via the TCR. Moreover, they make much less interferon-gamma when immunized with Toxoplasma gondii. Therefore, the alternative p38 activation pathway is important for normal T cell proliferation and immune/inflammatory responses. We have established two animal models that allow us to explore the importance of the alternative pathway in normal and pathophysiologic conditions. The findings include: - The p38alpha YF knockin mice have been crossed with the GADD45a knockout mice, and we found that the T cell hyperproliferation and autoimmunity was prevented. This establishes TCR-induced p38 activation as having a key role in autoimmune vasculitis. - We have made p38beta YF knockin mice and have crossed them to the p38alpha YF knockin mice (p38abYF). Quantitation of activated p38 in the various mutant animals allowed us to determine that p38alpha contributes about 70%, and p38beta about 30%, to total p38 activation via TCR signaling. The double knockin mice have more pronounced defects in T cell proliferation than mice with single mutations. Moreover, Th1 skewing is much reduced in cells from these animals, consistent with a decrease in sustained Tbet expression. These observations prove that p38alpha and p38beta are partly redundant, and that both need to be mutated to quantitate the importance of TCR-induced p38 activity in biological responses. - The p38abYF mice are significantly more resistant to two models of autoimmunity and inflammation: collagen-induced arthritis and experimental autoimmune encephalomyelitis. The former, in particular, is remarkably milder in the mutant mice, which had no evidence of severe joint involvement, whereas 75% or more of wild type mice developed ankylosis. - T cells from p38abYF mice file to upregulate critical transcription factors involved in Th17 skewing (NFATc1, IRF4) and, as a consequence, IL-17. - p38abYF mice mount a weak IL-17 response to Citrobacter and fail to clear the pathogen from the gut. - Activation of the classic MAPK pathway results in p38 activation that antagonizes the alternative pathway, inhibition T cell functional responses. - The molecular mechanism of the antagonism is MAPK-activated p38 phosphorylation and inactivation of NFATc1. - Biopsies of human psoriatic lesions before and after UV treatment have shown that MAPK UV-activated p38 inactivates NFATc1, leading to downregulation of IRF4 and IL-17. This is the first evidence that phototherapy acts on pathogenic T cells. - Membrane-permeable Gadd45a, but not an internal deletion mutant that can't bind p38, inhibits T cell proliferation and cytokine production by WT T cells. - Intratumor injection of this compound into Panc02 pancreatic tumors halts tumor growth and inhibits proinflammatory cytokine production by tumor infiltrating CD4+ T cells (TIL). - I.V. injection of the peptide into KPC mice (a mouse model of human pancreatic cancer) inhibits the progression of pre-neoplastic to neoplastic lesions. Just as with Panc02, proinflammatory cytokine production by TIL is reduced. - Examination of 193 human pancreatic cancers revealed that all have CD4+ TIL with alternatively activated p38. Furthermore, the was a highly statistically significant correlation between the percent of TIL with alternatively activated p38 and disease severity. These T cells secreted TNF and IL-17, just like their muring counterparts. - High throughput screens have identified a family of small molecules that are cell permeable, non-toxic, and inhibit the alternative p38 activation pathway. Studies in animal models of inflammation will be performed. Our observations establish the alternative p38 pathway as the only mechanism for the activation of this important kinase upon TCR-mediated stimulation, and show that TCR-activated p38 plays a critical role in several mouse models of human disease, and is highly active in human pancreatic cancer. The data support the importance of the alternative p38 activation pathway as a molecular target.